Combinations for the treatment of inflammatory disorders

ABSTRACT

The invention features a pharmaceutical composition that includes amoxapine and a corticosteroid.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Utility ApplicationSer. No. 10/191,149, filed Jul. 9, 2002, which claims the benefit ofU.S. Provisional Application Serial No. 60/304,089, filed Jul. 9, 2001,each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The invention relates to the treatment of inflammatory disorders.

[0003] Inflammation occurs when tissues are injured by viruses,bacteria, trauma, chemicals, heat, cold or any other harmful stimulus.Chemicals including bradykinin, histamine, serotonin and others arereleased, attracting tissue macrophages and white blood cells tolocalize in an area to engulf and destroy foreign substances. Duringthis process, chemical mediators such as TNFα are released, giving riseto inflammation. Inflammatory disorders are those in which theinflammation is sustained or chronic. One example of an inflammatorydisorder is osteoarthritis.

[0004] Immunoinflammatory disorders (e.g., rheumatoid arthritis,psoriasis, ulcerative colitis, Crohn's disease, stroke-induced braincell death, ankylosing spondylitis, fibromyalgia, and autoimmunediseases such as asthma, multiple sclerosis, type I diabetes, systemiclupus erythematosus, scleroderma, systemic sclerosis, and Sjögren'ssyndrome) are inflammatory disorders characterized by dysregulation ofthe immune system and inappropriate mobilization of body's defensesagainst its own healthy tissue.

[0005] One percent of humans world-wide are afflicted with rheumatoidarthritis, a relentless, progressive disease causing severe swelling,pain, and eventual deformity and destruction of joints. According to theArthritis Foundation, rheumatoid arthritis currently affects over twomillion Americans, of which women are three times more likely to beafflicted. Rheumatoid arthritis is characterized by inflammation of thelining of the joints and/or other internal organs, and the presence ofelevated numbers of lymphocytes and high levels of proinflammatorycytokines.

[0006] Treatment of rheumatoid arthritis generally includesadministration of (i) non-steroidal anti-inflammatory drugs (NSAIDs;e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate,mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin,piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, cholinesalicylate, salsalte, and sodium and magnesium salicylate); (ii)steroids (e.g., cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone); (iii)DMARDs, i.e., disease modifying antirheumatic drugs (e.g., cyclosporine,azathioprine, methotrexate, leflunomide, cyclophosphamide,hydroxychloroquine, sulfasalazine, D-penicillamine, minocycline, andgold); or (iv) recombinant proteins (e.g., ENBREL® (etanercept, asoluble TNF receptor) and REMICADE® (infliximab) a chimeric monoclonalanti-TNF antibody).

[0007] There is a need to develop new regimens for the treatment ofrheumatoid arthritis, and other inflammatory disorders.

SUMMARY OF THE INVENTION

[0008] We have discovered that the combination of amoxapine(2-cloro-11(1-piperazinyl)dibenz[b,f][1,4]oxapine) and prednisolone(also known as 1-dehydrocortisol; 1-dehydrohydrocortisone;1,4-pregnadiene-11beta,17alpha,21-triol-3,20-dione; and11beta,17alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione) brings aboutsubstantial suppression of TNFα levels induced in peripheral bloodmononuclear cells (PBMCs).

[0009] Amoxapine is a tricyclic antidepressant (TCA). Based on theability of amoxapine to act in concert with prednisolone to inhibit TNFαlevels, one skilled in the art will recognize that other TCAs can alsobe used in the present invention. Structural analogs of amoxapine thatare not tricyclic antidepressants can also be used. Exemplary structuralanalogs include, for example, clothiapine, perlapine, fluperlapine, anddibenz (b,f)(1,4)oxazepine, 2-chloro-11-(4-methyl-1-piperazinyl)-,monohydrochloride.

[0010] Prednisolone is a corticosteroid. Based on the shared structuralfeatures and apparent mechanism of action among the corticosteroidfamily, one skilled in the art will recognize that other corticosteroidscan be used in combination with amoxapine or other TCAs to treatinflammatory disorders.

[0011] Accordingly, the invention features a method for treating apatient having an inflammatory disorder, by administering to the patient(i) a TCA (e.g, amoxapine); and (ii) a corticosteroid (e.g.,prednisolone) simultaneously or within 14 days of each other, in amountssufficient to suppress TNFα levels sufficiently to produce a therapeuticbenefit to the patient. In one embodiment, the two compounds areamoxapine and prednisolone.

[0012] The invention also features a method for treating a patienthaving an inflammatory disorder by administering to the patient (i)clothiapine, perlapine, fluperlapine, or dibenz (b,f)(1,4)oxazepine,2-chloro-11-(4-methyl-1-piperazinyl)-, monohydrochloride; and (ii) acorticosteroid (e.g., prednisolone) simultaneously or within 14 days ofeach other, in amounts sufficient to suppress TNFα levels sufficientlyto produce a therapeutic benefit to the patient.

[0013] Preferably, the two compounds of the invention are administeredwithin ten days of each other, more preferably within five days of eachother, and most preferably within twenty-four hours of each other, orsimultaneously. The disorder treated according to the invention can be,for example, rheumatoid arthritis, psoriasis, ulcerative colitis,Crohn's disease, stroke-induced brain cell death, ankylosingspondylitis, and fibromyalgia, asthma, multiple sclerosis, type Idiabetes, systemic lupus erythematosus, scleroderma, systemic sclerosis,or Sjögren's syndrome.

[0014] In the above-described treatment method, both compounds arepreferably provided together in a pharmaceutical composition that alsoincludes a pharmaceutically acceptable carrier.

[0015] The invention also features a pharmaceutical composition thatincludes (i) a TCA (e.g., amoxapine); and (ii) a corticosteroid (e.g.,prednisolone), along with a pharmaceutically acceptable carrier,diluent, or excipient.

[0016] The invention also features a method for identifying compoundsuseful for treating a patient having an inflammatory disorder. Themethod includes the steps of: contacting immune cells in vitro with (i)a TCA or a corticosteroid; and (ii) a candidate compound, anddetermining whether the immune response is modulated relative to (a)immune cells contacted with the TCA or corticosteroid but not contactedwith the candidate compound, and (b) immune cells contacted with thecandidate compound but not with the TCA or corticosteroid. A candidatecompound that, when combined with the TCA or corticosteroid, modulatesthe immune response to a greater degree than controls, is a compoundthat is potentially useful for treating a patient having an inflammatorydisorder.

[0017] Compounds useful in the invention include those described hereinin any of their pharmaceutically acceptable forms, including isomerssuch as diastereomers and enantiomers, salts, solvates, and polymorphsthereof, as well as racemic mixtures of the compounds described herein.

[0018] By “tricyclic antidepressant” or “TCA” is meant a compound havingone the formulas (I), (II), or (III):

[0019] wherein each X is, independently, H, Cl, F, Br, I, CH₃, CF₃, OH,OCH₃, CH₂CH₃, or OCH₂CH₃;Y is CH₂, O, NH, S(O)₀₋₂, (CH₂)₃, (CH)₂, CH₂O,CH₂NH, CHN, or CH₂S; Z is C or S; A is a branched or unbranched,saturated or monounsaturated hydrocarbon chain having between 3 and 6carbons, inclusive; each B is, independently, H, Cl, F, Br, I, CX₃,CH₂CH₃, OCX₃, or OCX₂CX₃; and D is CH₂, O, NH, S(O)₀₋₂.

[0020] In preferred embodiments, each X is, independently, H, Cl, or F;Y is (CH₂)₂, Z is C; A is (CH₂)₃; and each B is, independently, H, Cl,or F.

[0021] Exemplary tricyclic antidepressants include, for example,amoxapine, 8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine, loxapinesuccinate, loxapine hydrochloride, 8-hydroxyloxapine, amitriptyline,clopramine, doxepin, imipramine, trimipramine, desipramine,nortriptyline, and protriptyline.

[0022] By “corticosteroid” is meant any naturally occurring or syntheticsteroid hormone which can be derived from cholesterol and ischaracterized by a hydrogenated cyclopentanoperhydrophenanthrene ringsystem. Naturally occurring corticosteriods are generally produced bythe adrenal cortex. Synthetic corticosteriods may be halogenated.Functional groups required for activity include a double bond at Δ4, aC3 ketone, and a C20 ketone. Corticosteroids may have glucocorticoidand/or mineralocorticoid activity.

[0023] Exemplary corticosteroids include, for example, dexamethasone,betamethasone, triamcinolone, triamcinolone acetonide, triamcinolonediacetate, triamcinolone hexacetonide, beclomethasone, dipropionate,beclomethasone dipropionate monohydrate, flumethasone pivalate,diflorasone diacetate, fluocinolone acetonide, fluorometholone,fluorometholone acetate, clobetasol propionate, desoximethasone,fluoxymesterone, fluprednisolone, hydrocortisone, hydrocortisoneacetate, hydrocortisone butyrate, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, hydrocortisone cypionate,hydrocortisone probutate, hydrocortisone valerate, cortisone acetate,paramethasone acetate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, prednisolone, prednisolone acetate,prednisolone sodium phosphate, prednisolone tebutate, clocortolonepivalate, flucinolone, dexamethasone 21-acetate, betamethasone17-valerate, isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone,dichlorisone, meclorisone, flupredidene, doxibetasol, halopredone,halometasone, clobetasone, diflucortolone, isoflupredone acetate,fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone,fluocinolone, clobetasol, cortisone, paramethasone, clocortolone,prednisolone 21-hemisuccinate free acid, prednisolonemetasulphobenzoate, prednisolone terbutate, and triamcinolone acetonide21-palmitate.

[0024] By “low dose corticosteroid” is meant a dose that is less than adose that would typically be given to a patient for treatment ofinflammation. Exemplary low doses of corticosteroids are as follows:cortisol: 12 mg/day; cortisone: 15 mg/day; prednisone: 3 mg/day;methylprednisolone: 2.5 mg/day; triamcinolone: 2.5 mg/day;betamethasone: 250 μg/day; dexamethasone: 450 μg/day; hydrocortisone: 9mg/day.

[0025] By a “dosage equivalent to an amoxapine dosage” is meant a dosageof a TCA that, in combination with a given dosage of prednisolone,produces the same anti-inflammatory effect in a patient as a dosage ofamoxapine in combination with that dosage of prednisolone.

[0026] By a “dosage equivalent to a prednisolone dosage” is meant adosage of a corticosteroid that, in combination with a given dosage ofamoxapine, produces the same anti-inflammatory effect in a patient as adosage of prednisolone in combination with that dosage of amoxapine.

[0027] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DETAILED DESCRIPTION

[0028] We have discovered that the combination of amoxapine andprednisolone exhibit substantial TNFα suppressing activity againstPBMCs. Concentrations that exhibited effective TNFα suppressing activitywere not unacceptably toxic to normal cells. Thus, this drug combinationis useful for the treatment of an inflammatory disorder.

Amoxapine

[0029] Amoxapine is a tricyclic antidepressant (TCA) of thedibenzoxapine type. It is structurally similar to the older TCAs andalso shares similarities with the phenothiazines.

[0030] The exact action of TCAs is not fully understood, but it isbelieved that one of their important effects is the enhancement of theactions of norepinephrine and serotonin by blocking the reuptake ofvarious neurotransmitters at the neuronal membrane. Amoxapine alsoshares some similarity with antipsychotic drugs in that it blocksdopamine receptors and can cause dyskinesia. Amoxapine also blocks thereuptake of norepinephrine, similar to the action of desipramine andmaprotiline.

[0031] Based on the ability of amoxapine to act in concert withprednisolone to inhibit TNFα levels, one skilled in the art willrecognize that other TCAs, as well as structural and functional analogsof amoxapine, can also be used in combination with prednisolone (oranother corticosteroid—see below). Amoxapine analogs include, forexample, 8-hydroxyamoxapine, 7-hydroxyamoxapine, loxapine, loxapinesuccinate, loxapine hydrochloride, 8-hydroxyloxapine, clothiapine,perlapine, fluperlapine, and dibenz (b,f)(1,4)oxazepine,2-chloro-11-(4-methyl-1-piperazinyl)-, monohydrochloride.

Prednisolone

[0032] Prednisolone, a synthetic adrenal corticosteroid, hasanti-inflammatory properties, and is used in a wide variety ofinflammatory conditions. It is desirable to reduce the amount ofadministered prednisolone because long-term use of steroids at canproduce significant side effects.

[0033] Prednisolone is a member of the corticosteroid family ofsteroids. Based on the shared structural features and apparent mechanismof action among the corticosteroid family, one skilled in the art willrecognize that other corticosteroids can be used in combination withamoxapine or an amoxapine analog to treat inflammatory disorders.Corticosteroids include, for example, dexamethasone, betamethasone,triamcinolone, triamcinolone acetonide, triamcinolone diacetate,triamcinolone hexacetonide, beclomethasone, dipropionate, beclomethasonedipropionate monohydrate, flumethasone pivalate, diflorasone diacetate,fluocinolone acetonide, fluorometholone, fluorometholone acetate,clobetasol propionate, desoximethasone, fluoxymesterone,fluprednisolone, hydrocortisone, hydrocortisone acetate, hydrocortisonebutyrate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, hydrocortisone cypionate, hydrocortisone probutate,hydrocortisone valerate, cortisone acetate, paramethasone acetate,methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, prednisolone, prednisolone acetate, prednisolonesodium phosphate, prednisolone tebutate, clocortolone pivalate,flucinolone, dexamethasone 21-acetate, betamethasone 17-valerate,isoflupredone, 9-fluorocortisone, 6-hydroxydexamethasone, dichlorisone,meclorisone, flupredidene, doxibetasol, halopredone, halometasone,clobetasone, diflucortolone, isoflupredone acetate,fluorohydroxyandrostenedione, beclomethasone, flumethasone, diflorasone,fluocinolone, clobetasol, cortisone, paramethasone, clocortolone,prednisolone 21-hemisuccinate free acid, prednisolonemetasulphobenzoate, prednisolone terbutate, and triamcinolone acetonide21-palmitate.

Therapy

[0034] Combination therapy according to the invention may be performedalone or in conjunction with another therapy and may be provided athome, the doctor's office, a clinic, a hospital's outpatient department,or a hospital. Treatment generally begins at a hospital so that thedoctor can observe the therapy's effects closely and make anyadjustments that are needed. The duration of the combination therapydepends on the type of inflammatory disorder being treated, the age andcondition of the patient, the stage and type of the patient's disease,and how the patient responds to the treatment. Additionally, a personhaving a greater risk of developing an inflammatory disorder (e.g., aperson who is genetically predisposed or previously had an inflammatorydisorder) may receive prophylactic treatment to inhibit or delay aninflammatory response.

[0035] The dosage, frequency and mode of administration of eachcomponent of the combination can be controlled independently. Forexample, one compound may be administered orally three times per day,while the second compound may be administered intramuscularly once perday. Combination therapy may be given in on-and-off cycles that includerest periods. The compounds may also be formulated together such thatone administration delivers both compounds.

Formulation of Pharmaceutical Compositions

[0036] Suitable modes of administration include oral, rectal,intravenous, intramuscular, subcutaneous, inhalation, topical ortransdermal, vaginal, and ophthalmic.

[0037] The combination of the invention can also be provided ascomponents of a pharmaceutical pack. The two drugs can be formulatedtogether or separately and in individual dosage amounts.

[0038] The compounds of the invention are also useful when formulated assalts. For example, amitriptyline, another tricyclic antidepressant, hasbeen formulated as a hydrochloride salt, indicating that amoxapine canbe similarly formulated.

[0039] Administration of each compound of the combination may be by anysuitable means that results in a concentration of the compound that,combined with the other compound, is anti-inflammatory. Each compound isadmixed with a suitable carrier substance, and is generally present inan amount of 1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable for oral,parenteral (e.g., intravenous, intramuscular, subcutaneous), rectal,transdermal, nasal, vaginal, inhalant, or ocular administration. Thus,the composition may be in form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, deliverydevices, suppositories, enemas, injectables, implants, sprays, oraerosols. The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, (19th ed.) ed. A. R. Gennaro, 1995, MackPublishing Company, Easton, Pa. and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, N.Y.

[0040] Pharmaceutical compositions according to the invention may beformulated to release the active compound substantially immediately uponadministration or at any predetermined time period after administration,using controlled release formulations.

[0041] Administration of compounds in controlled release formulations isuseful where the compound, either alone or in combination, has (i) anarrow therapeutic index (e.g., the difference between the plasmaconcentration leading to harmful side effects or toxic reactions and theplasma concentration leading to a therapeutic effect is small;generally, the therapeutic index, TI, is defined as the ratio of medianlethal dose (LD₅₀) to median effective dose (ED₅₀)); (ii) a narrowabsorption window in the gastro-intestinal tract; or (iii) a shortbiological half-life, so that frequent dosing during a day is requiredin order to sustain the plasma level at a therapeutic level.

[0042] Many strategies can be pursued to obtain controlled release inwhich the rate of release outweighs the rate of metabolism of thetherapeutic compound. For example, controlled release can be obtained bythe appropriate selection of formulation parameters and ingredients,including, e.g., appropriate controlled release compositions andcoatings. Examples include single or multiple unit tablet or capsulecompositions, oil solutions, suspensions, emulsions, microcapsules,microspheres, nanoparticles, patches, and liposomes.

[0043] Solid Dosage Forms for Oral Use

[0044] Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

[0045] The two compounds may be mixed together in a tablet or othervehicle, or may be partitioned. In one example, the first compound iscontained on the inside of the tablet, and the second compound is on theoutside, such that a substantial portion of the second compound isreleased prior to the release of the first compound.

[0046] Formulations for oral use may also be provided as chewabletablets, or as hard gelatin capsules wherein the active ingredient ismixed with an inert solid diluent, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium.

Dosages

[0047] The dosage of each compound of the claimed combinations dependson several factors, including: the administration method, the disease tobe treated, the severity of the disease, whether the disease is to betreated or prevented, and the age, weight, and health of the person tobe treated. Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular patient may affect dosage used.

[0048] Continuous daily dosing with the combinations of the inventionmay not be required. A therapeutic regimen may require cycles, duringwhich time a drug is not administered, or therapy may be provided on anas needed basis during periods of acute inflammation.

[0049] As described above, the compound in question may be administeredorally in the form of tablets, capsules, elixirs or syrups, or rectallyin the form of suppositories. Parenteral administration of a compound issuitably performed, for example, in the form of saline solutions or withthe compound incorporated into liposomes. In cases where the compound initself is not sufficiently soluble to be dissolved, a solubilizer suchas ethanol can be applied.

[0050] Below, for illustrative purposes, the dosages for amoxapine andprednisolone are described. One skilled in the art will readily be ableto ascertain suitable dosages for other TCAs and corticosteroids. Forexample, a TCA can be given in a dosage equivalent to an amoxapinedosage provided below, and a corticosteroid can be given in a dosageequivalent to a predinisolone dosage provided below. In one embodiment,the corticosteroid is a low dose corticosteroid.

[0051] Oral Administration

[0052] For amoxapine adapted for oral administration for systemic use,the total daily dosage is normally about 1-600 mg (0.01-8.5 mg/kg),preferably about 25-400 mg (0.35-5.7 mg/kg), and more preferably about100-300 mg (1.4-4.2 mg/kg) total daily dose. Administration can be oneto three times daily for one day to one year, and may even be for thelife of the patient. Chronic, long-term administration will be indicatedin many cases. Daily dosages up to 600 mg may be necessary.

[0053] For prednisolone adapted for oral administration for systemicuse, the daily dosage is normally about 0.05-200 mg (0.7-2800 mcg/kg),preferably about 0.1-60 mg (1-850 mcg/kg), and more preferably about0.1-5 mg (4-70 mcg/kg). Because of the enhancing effect exhibited byamoxapine on prednisolone anti-inflammatory activity, low dosages ofprednisolone (e.g., 0.2, 0.4, 0.6, 0.8, 1, 2, or 3 mg/day), whencombined with a TCA, can be effective in treating inflammation.Administration one to four times daily is desirable. Like amoxapine,prednisilone may be administered for one day to one year, and may evenbe for the life of the patient. Dosages up to 200 mg per day may benecessary.

[0054] Rectal Administration

[0055] For compositions adapted for rectal use for preventing disease, asomewhat higher amount of a compound is usually preferred. Thus a totaldaily dosage of amoxapine is normally about 1-600 mg (0.01-8.5 mg/kg).Rectal administration of amoxapine is normally one to three times daily.A total daily dosage of prednisolone is normally about 0.1-100 mg(1-1420 mcg/kg). Rectal administration of prednisolone is normally oneto four times daily.

[0056] Intravenous Administration

[0057] For intravenous administration of amoxapine, a total daily dosageis about 1-400 mg (0.014-5.7 mg/kg), preferably about 10-200 mg(0.14-2.8 mg/kg) and more preferably about 25-100 mg (0.35-1.4 mg/kg).Intravenous administration of amoxapine is normally one to four timesdaily, but can be continuously infused.

[0058] For intravenous administration of prednisolone, a total dailydosage is about 0.05-200 mg (0.0007-2.8 mg/kg), preferably about 0.1-60mg (0.001-0.85 mg/kg), and more preferably about 0.1-5 mg (4-70 mcg/kg).Low dosages of prednisolone, described above, are most preferred.Intravenous administration of prednisolone is normally one to four timesdaily, but, like amoxapine, can be continuously infused.

[0059] Additional Routes of Administration

[0060] For intramuscular, subcutaneous, inhalation, topical, vaginal, orophthalmic administration of amoxapine, a total daily dosage is about1-400 mg (0.014-5.7 mg/kg), preferably about 10-200 mg (0.14-2.8 mg/kg),and more preferably about 25-100 mg (0.35-1.4 mg/kg), and a total dailydosage of prednisolone is about 0.1-100 mg (0.0014-1.42 mg/kg). By theseroutes, administration of each of amoxapine and prednisolone is,independently, one to four times daily.

[0061] The following examples are to illustrate the invention. They arenot meant to limit the invention in any way.

EXAMPLE 1 Preparation of Pairwise Compound Mixed Combination SerialDilution Matrix

[0062] Stock solutions of amoxapine (16 mg/ml) (Sigma-Aldrich, St.Louis, Mo.; catalog number A129) and prednisolone (1.6 mg/ml)(Sigma-Aldrich, catalog number P6004) were made in dimethylsulfoxide(DMSO). The amoxapine master plates were made by adding 25 μl of theconcentrated stock solution to columns 3, 9, and 15 (rows C through N)of a polypropylene 384-well storage plate that had been pre-filled with75 μl of anhydrous DMSO. Using a TomTec Quadra Plus liquid handler, the25 μl of amoxapine stock solution was serially diluted two-fold fourtimes into the adjacent columns (columns 4-7, 10-13, 16-19). The sixthcolumn (8, 14, and 20) did not receive any compound and served as avehicle control. The prednisolone master plates were made by adding 25μl of the concentrated prednisolone stock solution to the appropriatewells (row C, columns 3-8; row C, columns 9-14; row C, columns 15-20;row I, columns 3-8; row I, columns 9-14; row I, columns 15-20) of theappropriate prednisolone master polypropylene 384-well storage plate.These master plates had been pre-filled with 75 μl of anhydrous DMSO.Using the TomTec Quadra Plus liquid handler, the 25 μl was seriallydiluted two-fold four times in the adjacent rows (rows D-G, and J-M).The sixth row (H and N) did not receive any compound to serve as avehicle control. Master plates were sealed and stored at −20° C. untilready for use.

[0063] The final amoxapine/prednisolone combination plate was generatedby transferring 1 μl from each of the amoxapine and prednisolone masterplates to a dilution plate containing 100 μl of media (RPMI; Gibco BRL,#11875-085), 10% Fetal Bovine Serum (Gibco BRL, #25140-097), 2%Penicillin/Streptomycin (Gibco BRL, #15140-122)) using the TomTec QuadraPlus liquid handler. This dilution plate was then mixed and a 10 μlaliquot transferred to the final assay plate, which had been pre-filledwith 40 μl/well RPMI media containing the appropriate stimulant toactivate TNFα secretion (see below).

EXAMPLE 2 Assay for TNFα Suppressing Activity of Amoxapine andPrednisolone

[0064] The compound dilution matrix was assayed using a TNFα ELISAmethod. Briefly, a 100 μl suspension of diluted human peripheral bloodmononuclear cells (PBMCs) contained within each well of a polystyrene384-well plate (NalgeNunc) was stimulated to secrete TNFα by treatmentwith a final concentration of 10 ng/ml phorbol 12-myristate 13-acetate(Sigma) and 750 ng/μl ionomycin (Sigma). Various concentrations of eachtest compound were added at the time of stimulation. After 16-18 hoursof incubation at 37° C. in a humidified incubator, the plate wascentrifuged and the supernatant transferred to a white opaquepolystyrene 384 well plate (NalgeNunc, Maxisorb) coated with an anti-TNFantibody (PharMingen, #18631D). After a two-hour incubation, the platewas washed (Tecan PowerWasher 384) with phosphate buffered saline (PBS)containing 0.1 % Tween 20 (polyoxyethylene sorbitan monolaurate) andincubated for an additional one hour with another anti-TNF antibody thatwas biotin labeled (PharMingen, 18642D) and horseradish peroxidase (HRP)coupled to strepavidin (PharMingen, #13047E). After the plate was washedwith 0.1% Tween 20/PBS, the HRP substrate (which contains luminol,hydrogen peroxide, and an enhancer such as para-iodophenol) was added toeach well and light intensity measured using a LJL Analyst luminometer.Control wells contained a final concentration of 1 μg/ml cyclosporin A(Sigma).

EXAMPLE 3 Suppression of TNFα Secretion from Phorbol 12-myristate13-acetate and Ionomycin Treated Blood by a Combination of Amoxapine andPrednisolone

[0065] Together, amoxapine and prednisolone were able to suppressphorbol 12-myristate 13-acetate and ionomycin induced TNFα secretion inblood. As is shown in Tables 1 and 2, amoxapine is able to enhance thedose response of prednisolone by nearly two fold. At a concentration of1.11 μM, prednisolone alone is able to inhibit TNFα secretion by 28%.Addition of 0.2 μM amoxapine increases the TNFα inhibition of the 1.11μM prednisolone to 51%. This large increase in activity of 82% iscreated by a relatively small increase of only 18% in total drugspecies. TABLE 1 Amoxapine [μM] Prednisolone [μM] 12.750 3.188 0.7970.199 0.050 0.000 1.110 85.89 66.47 47.73 50.93 32.79 27.59 0.277 82.8258.88 48.79 34.40 29.17 26.81 0.069 78.58 60.66 41.32 34.99 21.41 22.310.017 84.69 62.66 34.66 32.48 21.39 19.06 0.004 84.13 53.41 33.86 16.025.82 2.69 0.000 73.02 50.44 24.29 16.66 7.30 0.00

[0066] TABLE 2 Amoxapine [μM] Prednisilone [μM] 12.747 6.373 3.187 1.5930.797 0.398 0.199 0.100 0.050 0.000 1.110 88.35 74.63 66.76 65.79 57.7252.50 45.50 43.72 40.04 34.62 0.555 90.09 76.42 69.66 60.08 53.51 46.7341.70 43.67 31.76 30.50 0.277 86.67 75.34 66.45 59.64 54.23 46.97 45.3835.20 34.42 36.89 0.139 91.50 78.45 70.37 60.75 54.73 47.05 41.51 37.7928.46 25.74 0.069 36.59 86.03 77.74 67.81 57.14 49.96 37.24 33.39 31.7524.56 0.035 92.76 80.28 70.42 59.40 52.58 47.40 37.94 34.59 21.47 24.060.017 91.02 75.16 72.06 56.40 45.14 47.84 36.50 24.33 21.92 24.74 0.00988.58 72.16 71.61 56.03 49.80 39.87 28.66 27.23 17.78 14.34 0.004 84.3266.14 57.21 46.53 32.06 27.76 23.73 15.94 12.99 9.62 0.000 80.89 64.4047.96 37.13 21.88 16.38 14.19 3.60 −3.31 −0.97

[0067] Amoxapine enhancement of prednisolone activity was also observedin a follow-up secondary screen. The TNFα inhibition of prednisolone ata concentration of 9 nM was increased 2.9 fold to 40% in the presence of400 nM amoxapine. The TNFα inhibition activity of prednisolone andamoxapine alone at these concentrations is only 14 and 16% respectively.Moreover, the level of TNFα inhibition achieved by 9 nM prednisolone incombination with 398 nM amoxapine (40%) is no less than that of 1110 nMprednisolone alone (35%). This increase in TNFα inhibition constitutes apotency shift of as much as 100-fold for the combination, compared toprednisolone alone.

[0068] The ability of amoxapine and prednisolone to inhibit TNFαsecretion from LPS stimulated blood is shown in Table 3. TABLE 3Amoxapine [μM] Prednisolone [μM] 12.747 6.373 3.187 1.593 0.797 0.3980.199 0.100 0.050 0.000 1.110 78.97 71.52 67.84 63.65 66.04 68.04 61.2964.30 58.19 60.06 0.555 83.61 68.05 62.72 65.82 59.46 56.17 56.36 55.4755.94 47.15 0.277 70.40 64.01 62.08 57.91 55.42 54.64 56.94 51.39 50.0548.75 0.139 72.56 60.77 58.36 55.47 50.42 49.25 49.54 48.74 44.46 48.460.069 70.27 73.99 61.88 48.82 43.56 47.22 42.13 42.62 44.19 38.79 0.03586.37 64.17 43.28 38.16 37.26 37.96 38.06 40.83 32.87 33.11 0.017 78.8148.94 42.94 40.81 37.94 35.96 32.00 35.25 29.35 37.12 0.009 67.09 43.7641.07 34.23 25.54 24.86 31.12 23.57 27.36 30.24 0.004 60.14 37.59 34.0325.52 24.94 27.78 25.57 30.40 18.90 12.06 0.000 49.64 21.26 24.21 16.7913.11 8.10 2.39 5.52 3.00 −1.31

Other Embodiments

[0069] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in molecular biology or related fields are intended to be withinthe scope of the invention.

What is claimed is:
 1. A pharmaceutical composition comprising amoxapineand a corticosteroid, wherein the amoxapine and corticosteroid arepresent in amounts that, when administered together to a patient havingan inflammatory disorder, inhibit or reduce inflammation.
 2. Thecomposition of claim 1, wherein amoxapine is present in the compositionin an amount of 1-600 milligrams.
 3. The composition of claim 2, whereinamoxapine is present in the composition in an amount of 100-300milligrams.
 4. The composition of claim 1, wherein the steroid ispresent in the composition in an amount of 0.05-200 milligrams.
 5. Thecomposition of claim 4, wherein the steroid is present in thecomposition in an amount of 0.05-5 milligrams.
 6. The composition ofclaim 5, wherein the steroid is present in the composition in an amountof 0.1-3 milligrams.
 7. The composition of claim 1, wherein thecomposition is formulated for oral administration.
 8. The composition ofclaim 1, wherein the composition is formulated for topicaladministration.